Commitment of common T/Natural killer (NK) progenitors to unipotent T and NK progenitors in the murine fetal thymus revealed by a single progenitor assay

Abstract

We have established a new clonal assay system that can evenly support the development of T and natural killer (NK) cells. With this system, we show that all T cell progenitors in the earliest CD44(+)CD25(-)FcgammaRII/III(-) fetal thymus (FT) cell population retain NK potential, and that the NK lineage-committed progenitors (p-NK) also exist in this population. T cell lineage-committed progenitors (p-T), which are unable to generate NK cells, first appear at the CD44(+)CD25(-) FcgammaRII/III(+) stage in day 12 FT. The proportion of p-T markedly increases during the transition from the CD44(+)CD25(-) stage to the CD44(+)CD25(+) stage in day 14 FT. On the other hand, p-NK preferentially increase in number at the CD44(+)CD25(-) stage between days 12 and 14 of gestation. The production of p-NK continues up to the CD44(+)CD25(+) stage, but ceases before the rearrangement of T cell receptor beta chain genes. It was further shown that the CD44(+)CD25(-) CD122(+) population of day 14 FT exclusively contains p-NK. These results indicate that the earliest T cell progenitor migrating into the FT is T/NK bipotent, and strongly suggest that the bipotent progenitor continuously produces p-NK and p-T until the CD44(+)CD25(+) stage.

Figure 1

In vitro generation of T and NK cells from FT progenitors. Unfractionated FT cells (10²) from 12-dpc B6Ly5.1 fetuses were cultured with a dGuo-treated FT lobe (B6) in the absence or presence of IL-2 (25 U/ml), IL-7 (50 U/ml), and SCF (10 ng/ml). At various days of culture, cells were collected from each well, counted, and analyzed by a flow cytometer. A and B show the kinetics of generation of CD3⁺NK1.1⁻ and CD3⁻NK1.1⁺ cells, respectively. (C) Representative flow cytometric profiles of cells recovered 10 d after culture for expression of NK1.1 vs. CD3, CD4 vs. CD8, and TCR-α/β vs. TCR-γ/δ on Ly5.1⁺ cells. Profiles of newborn thymus cells are shown for comparison.

Figure 1

In vitro generation of T and NK cells from FT progenitors. Unfractionated FT cells (10²) from 12-dpc B6Ly5.1 fetuses were cultured with a dGuo-treated FT lobe (B6) in the absence or presence of IL-2 (25 U/ml), IL-7 (50 U/ml), and SCF (10 ng/ml). At various days of culture, cells were collected from each well, counted, and analyzed by a flow cytometer. A and B show the kinetics of generation of CD3⁺NK1.1⁻ and CD3⁻NK1.1⁺ cells, respectively. (C) Representative flow cytometric profiles of cells recovered 10 d after culture for expression of NK1.1 vs. CD3, CD4 vs. CD8, and TCR-α/β vs. TCR-γ/δ on Ly5.1⁺ cells. Profiles of newborn thymus cells are shown for comparison.

Figure 1

In vitro generation of T and NK cells from FT progenitors. Unfractionated FT cells (10²) from 12-dpc B6Ly5.1 fetuses were cultured with a dGuo-treated FT lobe (B6) in the absence or presence of IL-2 (25 U/ml), IL-7 (50 U/ml), and SCF (10 ng/ml). At various days of culture, cells were collected from each well, counted, and analyzed by a flow cytometer. A and B show the kinetics of generation of CD3⁺NK1.1⁻ and CD3⁻NK1.1⁺ cells, respectively. (C) Representative flow cytometric profiles of cells recovered 10 d after culture for expression of NK1.1 vs. CD3, CD4 vs. CD8, and TCR-α/β vs. TCR-γ/δ on Ly5.1⁺ cells. Profiles of newborn thymus cells are shown for comparison.

Figure 2

Cytolytic activity of NK1.1⁺ cells derived from 12-dpc FT progenitors. Unfractionated FT cells (10²) from 12-dpc fetuses were cultured together with a dGuo-treated FT lobe in the presence of a cytokine cocktail (see legend to Fig. 1). After 10 d of culture, cells were recovered and pooled from three wells, then stained in three colors with anti-Ly5.1, anti-CD3, and anti-NK1.1. Ly5.1⁺CD3⁻NK1.1⁺ cells and Ly5.1⁺CD3⁻ NK1.1⁻ cells were obtained by FACS^® sorting, and were assayed for cytolytic activity against the target cell line Yac-1 as detailed in Materials and Methods.

Figure 3

Representative flow cytometric profiles of cells derived from single FT progenitors. (A) Procedure of the clonal assay culture is shown. Single CD44⁺CD25⁻ FT cells (14 dpc) were picked up under microscopical visualization, then seeded into the wells where a dGuo-treated FT lobe had been placed. The culture medium used was supplemented with a cytokine cocktail (see legend to Fig. 1). Cells were cultured under HOS conditions for 10 d. (B) Examples of the flow cytometric profiles of cells generated from p-NK, p-T, and p-T/NK are shown. Cell recoveries in these clones were 2.4 × 10⁴, 8.0 × 10⁴, and 2.0 × 10⁵, respectively.

Figure 4

Frequency and total number of different types of progenitors in subpopulations of 12- and 14-dpc FT cells. (A) Flow cytometric profiles of 12- and 14-dpc FT cells are shown. Numbers in panels represent percentage of cells. Cells in subpopulations gated with bars or rectangles in A served for the clonal assay (see Fig. 3 A). In all subpopulations, 50 individual cells were investigated. (B and C) The numbers of the different types of progenitors detected among 50 cells are scored (upper scales). In calculating the total number of progenitors in an FT (lower scales), the numbers of cells per 12-dpc FT and 14-dpc FT were regarded as 1,000 and 10,000, respectively.

Figure 5

NK cells retain their TCR-β and TCR-γ locus in the germline configuration. The Dβ2-Jβ2 gene rearrangement (A) and Vγ4/Vγ3-Jγ1 gene rearrangement (B) in cells derived from single p-T/NK, p-NK, and p-T were investigated. As a positive control, unfractionated adult thymocytes (AT) were used. The DNA equivalent to 750 cells was PCR amplified in each sample.

Figure 7

A model for NK and T cell lineage commitment in the murine FT. The T cell progenitors migrating into the FT are bipotent p-T/NK. Commitment of p-T/NK to p-NK occurs at a broad range of early differentiation stages, whereas commitment to p-T mainly occurs immediately before the TCR-β rearrangement. CD44⁺CD25⁻ cells in 12-dpc FT comprise FcR⁻ and FcR⁺ populations (see Fig. 4), whereas a majority of those in 14-dpc FT are FcR⁺ (reference 19). A portion of p-NK in 14-dpc FT express CD122. The width of the white arrows approximately correlates with the rate of p-T and p-NK generation. It is unclear whether p-NK also immigrate from extrathymic tissues (broken arrow).

Figure 6

The CD122⁺ population of FT cells contains exclusively p-NK. (A) Flow cytometric profiles of 14-dpc FT cells. (B) Single CD44⁺CD25⁻CD122⁻ or CD44⁺CD25⁻CD122⁺ cells (50 cells each) enclosed with rectangles in the right panel of A were cultured with a dGuo-treated FT lobe in the presence of the cytokine cocktail (see legend to Fig. 3). The numbers of the different types of progenitors detected among 50 CD122⁻ and 50 CD122⁺ cells are scored.